ABSTRACT RP-4 focuses on synovial sarcoma, an aggressive pediatric/young adult sarcoma driven by the SS18-SSX fusion oncogene. SS18-SSX has emerged as a multi-faceted disruptor of epigenetic control that mediates genome-wide transcriptional deregulation, resulting in proliferation and aberrant or arrested differentiation. Our overall goal is to probe the basic pathobiology of synovial sarcoma so as to nominate potential therapeutic targets. We propose hypothesis-driven screening approaches, namely functional genomic screens to uncover vulnerabilities among chromatin/transcriptional regulators and among kinases, as well as targeting the oncogenic fusion itself. Based on the hypothesis that SS18-SSX causes synovial sarcoma cells to possess special ?epigenetic? dependencies, we will, in Aim 1, (a) perform a broad functional genomic screen using a new CRISPR knock-out pooled library against epigenetic modulators; and (b) seek to further validate and understand mechanistically KDM2B, a dependency identified in preliminary studies, as a vulnerability in human synovial sarcoma cells. Based on the hypothesis that another effect of SS18-SSX is transcriptional deregulation of growth signaling, Aim 2 will employ three orthogonal strategies to better define targetable kinase vulnerabilities in this sarcoma. Specifically, we will (a) perform a CRISPR-based functional genomic screen against kinases in human synovial sarcoma cell lines; (b) identify activated kinases in synovial sarcoma by phospho-protein profiling of patient-derived xenografts; and (c) define the targets of pazopanib in synovial sarcoma by two complementary methods, affinity proteomics and PLATO (parallel analysis of translated ORFs). These two analyses should clarify the critical kinase targets of this multi-targeted kinase inhibitor that is active in a subset of synovial sarcoma patients. The results generated in this Aim will be integrated to form the basis for more rational targeting of critical kinases in this sarcoma. As SS18-SSX is the primary driver oncogene in synovial sarcoma, the junction point of the fusion transcript represents an rational and highly specific target for sequence-based therapeutics using new antisense oligonucleotide (ASO) approaches. Building on our promising results using gapmer ASOs to directly target other sarcoma fusions in vitro and in vivo, we will optimize and evaluate gapmer ASOs against SS18-SSX. In Aim 4, we will validate the targets identified in Aims 1-3 by confirming on-target effects and expression in human tumors, and we will perform preclinical evaluation of their potential as therapeutic targets in vitro and in vivo. The most promising targets will be validated and subjected to extensive preclinical evaluation using multiple, orthogonal, relevant in vitro and in vivo systems. The ultimate overall goal of this fundamentally translational project is to begin clinical evaluation of at least one agent based on the novel targets and strategies identified through our work in patients with this often lethal sarcoma within five years.